Recent outbreaks of highly pathogenic influenza virus have highlighted the need for a better understanding of the interactions between influenza virus and its host. Viral infection triggers a prompt anti-viral response, with type I interferons (IFNs) playing the central role in coordinating the host response through the upregulation of a large number of IFN stimulated genes (ISGs). Several ISGs have direct antiviral activity, while others impact upon the antiviral response by modulating the immune system. We have recently shown that one of these ISGs, ISG15, functions as a critical IFN induced anti-viral molecule. ISG15 is an ubiquitin homolog that is strongly upregulated by IFNs, toll receptor ligation, and viral infection. ISG15 deficient mice display increased lethality following infection with several viruses, including both influenza A and B viruses. Yet the mechanism by which ISG15 exerts this antiviral activity is unknown. ISG15 conjugates to a wide array of intracellular proteins, targeting numerous biological processes. Human ISG15 is also released from cells and functions as a cytokine, activating various immune cells. In this proposal we will test the hypothesis that the conjugation of ISG15 to target proteins results in the inhibition of viral replication within the respiratory epithelium and allows for subsequent clearance of the virus by the host. We will test this hypothesis with the following two aims. The studies in Aim 1 will explore the mechanism by which ISG15 regulates viral replication within the respiratory epithelium. We will also determine if expression of ISG15 within this cell type is sufficient to control influenza virus infection. The studies in Aim 2 will determine if conjugation of ISG15 is required for the antiviral activity of ISG15. We will determine if ISG15 conjugates to viral proteins and regulates viral replication. The results obtained from these studies will provide important insight into a potential mechanism of action for a newly identified antiviral molecule, ISG15. ISG15 functions as a critical antiviral molecule, with activity against several human pathogens, including both influenza A and B viruses. This proposal will explore its mechanism of action during influenza virus infection by investigating its sight of action and requirement for conjugation to target proteins. These studies may provide insight into a potential new therapeutic target in the fight against viral infections.